In
April 2002, Tyrone Hayes and his coworkers published astonishing
findings showing that atrazine causes tadpoles of the classic
"laboratory rat" of the frog world, the African clawed
toad Xenopus laevus, to become hermaphroditic as adults.

The
most remarkable aspect of this finding was the extraordinary low
levels at which the impact occurs, 0.1 parts per billion, an exposure
level not only astoundingly low but also one that is exceeded almost
ubiquitously in regions where atrazine is used. Indeed, as Hayes
et al. reported in that paper, rainwater away from regions
of atrazine use can sometimes carry 1 ppb... ten times the amount
they found sufficient to cause hermaphroditism in 20% of exposed
animals.

With
these two new papers, Hayes et al. answer affirmatively
two important questions raised by their earlier work: are other
species of amphibians similarly sensitive to atrazine, and can the
effects be observed in wild populations of frogs where atrazine
is used? Their new laboratory experiments demonstrate conclusively
that atrazine causes hermaphroditism in leopard frogs also.
And their field survey transecting the US from Iowa to Utah, reveals
hermaphroditic leopard frogs across a broad swath of the natural
distribution of this species, but only in areas where atrazine
can be detected (which is almost all places they surveyed).

What
did they do? Hayes et al. conducted a laboratory
experiment with leopard frogs and then carried out a field survey:

The
lab experiment: Hayes et al. exposed leopard frog
tadpoles to atrazine at two different levels in their water, 0.1
parts per billion and 25 ppb, and compared the percentage of animals
that matured as hermaphrodites in these treatments vs. a control
group that was not exposed to atrazine. Each exposure group contained
30 animals. The experiment was repeated completely three separate
times.

The
field survey: Hayes's field team sampled young leopard frogs
that had matured earlier that spring at a series of eight sites
across the US mid-west, from Iowa to Utah (see map below). The sites
were chosen based on USGS data on atrazine sales so that the survey
included areas of high and no or little atrazine use, while also
being within the natural range of the leopard frog. At each site,
they collected 100 animals, which they preserved and took back to
their laboratory at the Univ. of California, Berkeley, for dissection.

The
figure overlays a depiction of the natural distribution of leopard
frogs (light pink) within the US upon a map indicating the amount
of atrazine use (kilograms per square kilometer) based on within-county
atrazine sales. It also shows the location of 8 sites where Hayes
et al. collected frogs and water for analysis (numbers
1-8)

To
avoid expectation biases, Hayes' team used a double-blind system
that gave no clue as to the origin of the animal while before all
histological analysis was completed.

At
each site they also took water samples, which they sent to two different
laboratories for analysis to determine levels of atrazine and a
series of other pesticides reported to be used at or near the collection
sites. The labs were not told of the samples' origins. Limits of
atrazine detection of the labs analyses was 0.1 ppb.

What
did they find? The laboratory experiment revealed that
male leopard frogs are extremely sensitive to atrazine exposure
during metamorphosis from tadpole to adult.

Many
of the males exposed to both very low and low levels of atrazine
(0.1 and 25 ppb) had abnormalities in their reproductive tracts.
For example, 29% of the 0.1 ppb-treated animals and 8% of the animals
treated with 25 ppb displayed varying degrees of sex-reversal. No
cases of sex-reversal were seen in the controls. While only one
individual control animal out of all examined had under-developed
testes, 36% and 12% of the males treated with 0.1 and 25 ppb atrazine,
respectively, showed under-developed testes (gonadal dysgenesis).
This was seen in only 2 control animals. The exposed males that
had undergone almost complete sex-reversal had gonads almost completely
filled with oocytes. This was never seen in controls.

The
proportion of males leopard frogs with gonadal abnormalities,
comparing unexposed controls with animals treated with two
different levels of atrazine.

The
field surveys found reproductive abnormalities in wild leopard frogs
comparable to those seen in the experimentally-treated frogs, except
at the one site where measured atrazine levels were below 0.2 ppb,
where no abnormalities were found.

No
simple relationship between atrazine level measured and proportion
of gonadal abnormalities, except that abnormalities were found
everwhere except site 1, with the lowest atrazine level.

Hayes
et al. found no simple relationship between atrazine levels
and the likelihood of gonadal abnormalities, except that abnormalities
occurred everywhere except the site with the lowest atrazine leve
(site 1). The site with the highest level of measured atrazine (site
6) did not have the greatest proportion of abnormalities.

These
inconsistencies suggest several possible interpretations. Most importantly,
water sampling and frog collection occurred a month or more after
tadpole exposure to atrazine... the scientists had to wait until
after metamorphosis to find maturing individuals for dissection.
There is no way using Hayes's existing data to work backward from
the samples collected to determine actual exposure levels prior
to metamorphosis. The one site with extremely low atrazine levels
in an area with almost no atrazine use is the one site with no gonadal
abnormalities. All other sites had atrazine levels well above the
range shown by the experiments to induce abnormalities. All of these
sites had male frogs with gonadal abnormalities.

What
does it mean? The lab studies confirm that male gonadal
development in leopard frogs can be disrupted by extremely low levels
of atrazine. The field studies reveal widespread gonadal abnormalities
in regions where atrazine contamination is within the range shown
by the laboratory studies to disrupt development. This does not
prove with certainty that the effects observed in wild leopard frog
populations are caused by atrazine, but it is strong circumstantial
evidence. The case will be strengthened--or refuted--by further
studies that measure atrazine levels prior to metamorphosis, analogous
to the experimental design. The fact that atrazine contamination
is so widespread complicates the practicalities of executing this
design.

The
case is also complicated by the possibility that regular exposure
to atrazine over many frog generations may have led to development
of pesticide resistance. This hypothesis would predict that populations
in the regions of most prolonged and intense atrazine use should
have lower proportions of gonadal abnormalities than those with
lesser and shorter traditions of use. If atrazine use histories
can be established for different sites, this could be approached
experimentally through laboratory experiments with animals from
those sites.

Hayes'
work raises significant but unanswered questions about the role
of atrazine in contributing to frog population declines. As they
describe, the timing of atrazine use in crops coincides with the
period of tadpole growth revealed by the experiments to be sensitive
to atrazine exposure. The sites where tadpoles mature, moreover,
are often precisely in the drainage wetlands that are the principal
recipients of atrazine run-off from crops. Perhaps paradoxically,
Hayes et al. note that juvenile frogs were abundant at the study
sites. Clearly some reproduction is occurring. One possibility,
mentioned above, is that pesticide resistance has evolved.

Hayes
et al. conclude:

"There
are likely many factors involved in amphibian declines. Endocrine
disruption by pesticides is but one potential cause and atrazine
only one such compound. However, given the widespread use
and ubiquitous contamination by atrazine, its pattern of use,
and its potency as an endocrine disruptor, atrazine likely
has a significant impact on amphibian populations. In particular,
given recent evidence
that atrazine potentiates parasitic infections in amphibians
in addition to its impact on reproductive development, the
role of atrazine in amphibian declines is of particular concern.
Further, enhancement of atrazine effects when mixed with other
pesticides, as indicated in our ongoing studies, must be explored."